In a third presentation from Predictive Biosciences, researchers also described a technique developed to detect hypermethylated genes in bodily fluids such as urine. They have incorporated into the multi-analyte diagnostic readout assay described above a next-gen bisulfate sequencing approach to determine the methylation of CpG sites at single-base resolution.
Identification of altered methylation patterns is an increasingly valuable diagnostic tool in cancer. The technique described here “could be used to detect other hypermethylated genes in a variety of bodily fluids where percent methylation may be much lower than in tumor tissue samples,” concluded the authors. “By developing a version of the assay that integrates all of the biomarkers in a format that can be run on any NGS platform, we can increase throughput, reduce cost, and make the assay ready for in vitro diagnostic kit development,” explained Shuber.
Nicola Normanno, Centro Richerche Oncologiche Mercogliano (Italy), and an international team of colleagues that participate in the OncoNetwork Consortium, evaluated a custom gene panel that targets hotspot mutations in 22 genes implicated in colorectal cancer and non-small-cell lung carcinoma. The panel is intended for use in a clinical research setting to help classify colon and lung tumors based on the detection of cancer-associated gene mutations.
Normanno et al., described the three-phase study the consortium used to verify the panel using 155 FFPE tissue samples and controls on an Ion Torrent 316 chip.
Ten ng of sample were processed on the OncoNetwork panel following amplification with the Ion AmpliSeq Library Kit 2.0. The authors reported high average per base accuracy of the panel, detection of all expected variants, a 100% true positive rate for newly identified variants as confirmed using Sanger sequencing, and 100% reproducibility of results across six different testing laboratories. A second gene panel, designed to optimize read distribution over the amplicons, was introduced during the course of the three-phase verification study, and phases 1 and 2 of the study were repeated using the new panel design. It allowed for multiplexing of up to eight samples on a 316 chip with an average read depth of 2,750 and improved coverage uniformity. Repeat testing confirmed 100% reproducibility and high genotyping sensitivity of the new panel.
Todd Hembrough, Ph.D., and colleagues from OncoPlex Diagnostics and the University of Chicago described their work to develop a multiplexed mass spectrometry-based quantitative assay for colorectal cancer (CRC) using liquid tissue-selected reaction monitoring (SRM). The assay is based on an understanding of the relationship between overexpression of multiple oncogenes, including those encoding several families of receptor tyrosine kinases, and tumor heterogeneity.
This link allows for characterization of protein expression by targeted proteomics in CRC tumors to categorize patients into molecular subsets and identify a personalized treatment strategy. “Moreover, intra-patient tumor heterogeneity from primary tumor to metastatic disease is likely to influence biomarker prediction of response to specific targeted agents,” stated the researchers.
They validated the CRC-plex SRM assay preclinically on cell lines, and demonstrated a good correlation between Liquid Tissue®-SRM analysis and ELISA for quantification of selected oncogene proteins. In this study the authors measured oncogene protein levels using SRM analysis in 42 CRC-paired primary and metastatic tumor tissue samples from the same patients. They were able to measure as many as 20 target proteins in a multiplexed assay using FFPE tumor tissue samples.
A comparison of primary CRC tumors with their paired metastatic samples showed a statistically significant increase in cMet expression for metastatic vs. primary tissues, “suggesting that in the absence of gene amplification, cMet may be a druggable target in these tissues,” concluded the authors.
Leveraging Molecular Techniques
A team of researchers from MolecularMD was focused on PTEN, a tumor suppressor that exerts its effects via dephosphorylation. Loss of PTEN function has been associated with multiple cancers, including endometrial cancer, glioblastoma, melanoma, and prostate cancer. Loss of PTEN activity can result from more than one independent aberration, including mutations in the coding regions (exons) of the PTEN gene, genomic deletions, or promoter methylation, in which the PTEN gene may be present and unadulterated but epigenetic changes to the regulatory DNA upstream of the gene block expression, resulting in loss of the PTEN protein.
The researchers from MolecularMD evaluated FFPE tissue samples using three different methods, allowing them to assess PTEN status at both the DNA and protein levels. Both “may be of important for clinical decision-making because loss of PTEN is associated with resistance to anti-EGFR therapies,” they explained.
Using a Sanger sequencing assay on genomic DNA extracted from FFPE tissue sections and amplified with PCR they could detect known hot-spot mutations in specific exons. The automated immunohistochemistry assay they developed was validated in cell lines and FFPE tissues. If >10% of the tumor cells in a sample stained, the sample was defined as positive for PTEN protein. The two-color dual chromogenic and silver in situ hybridization (ISH) assay was scored as follows: >20% loss of PTEN signal identifies tumors having heterozygous loss of the PTEN gene; >30% loss of PTEN signal indicates homozygous loss of the PTEN gene.
Assessment of 22 FFPE tissue specimens showed that of the nine samples identified as wild-type by Sanger sequencing, seven had normal PTEN protein expression on IHC. The fact that two samples were IHC-negative suggests that mechanisms other than mutations in the coding regions of the PTEN gene are responsible for the loss of PTEN protein. Of the 13 samples found to have hot-spot mutations on Sanger sequencing, seven showed loss of PTEN protein on IHC; the other six samples had normal PTEN protein expression.
This finding also supports the observation that mutations in gene coding region hot-spots are not sufficient on their own to predict PTEN protein expression, and vice versa. The authors concluded that complementary molecular diagnostic methods that assess the mechanisms underlying changes in gene and protein expression are needed for accurate clinical assessment of PTEN status in FFPE tissue samples.